(1) Field of the Invention:
The present invention relates to a method of quantitatively measuring an oxidative substance while avoiding the influences of a coloring-interfering substance in the quantitative measurement of a component in a body fluid by using a triphenyl methane type leuco-pigment as a coloring reagent. The invention also relates to a method of quantitatively measuring an oxidative substance with coloring sensitivity thereof being adjustable in such a quantitative measurement of the body fluid component.
(2) Description of the Prior Art:
Triphenyl methane type leuco-pigment is superior to the conventional oxidizable coloring reagents in that when oxidatively colored, the pigment has a coloring wavelength of not less than 600 nm on the long wavelength side, and a large molecular absorption coefficient of not less than 50,000. In addition, the color does not fade over time after color development. Thus, they have been experimentally applied to the quantitative measurement of a very small amount of an oxidative substance such as hydrogen peroxide or a quantitative measurement of peroxidase-like substance, and the like. For instance, it is reported in Analytical Chemistry Vol. 42, p. 410-411 (1970) that H.sub.2p O.sub.2 can be quantitatively measured by using a combined reagent of leucocrystal violet (LCV) and peroxidase (POD). In Clinical Chemistry Vol. 21, p. 362-369 (1975), there are reported experimental results in the quantitative measurement of heme compounds such as hemoglobin and the like using leucomalachite green (LMG) and H.sub.2 O.sub.2. However, the quantitative measurement of H.sub.2 O.sub.2 in the former case is merely an ordinary H.sub.2 O.sub.2 quantitative measurement. It does not refer to the quantitative measurement of H.sub.2 O.sub.2 generated by an enzyme reaction in the quantitative measurement of the component in the body fluid where linearity lineality of a calibration curve is poor due to the influences of protein in samples. Meanwhile, Japanese Patent Application Laid Open No. 26,199/1981 discloses a method of quantitatively measuring a component present in a very small amount in serum, urine or the like using bis(p-diethylaminophenyl)-2-sulfophenyl methane (hereinafter abbreviated as BSPM), which belongs to the triphenyl methane type leuco coloring matters. According to the trace experiment of the present inventors, quantitative determinations, described in the specification of this publication, could not be obtained with serum. That is, the triphenyl methane type leuco-pigment has not seen practical use in the field of clinical chemical analysis.
On the other hand, a triphenyl methane type leuco coloring matter of a high sensitivity has been developed and its practical application is expected. Compared with the conventional oxidizable coloring reagent, this coloring reagent has the advantages that the coloring wavelength is on the long wavelength side, not less than 600 nm, the molecular absorption coefficient is not less than 70,000, and almost no color fading takes place with a lapse of time after color development. However, due to its high sensitivity, there may be difficulties in practice. That is, its use is limited to a narrow measuring range of the quantitative measurement of an extremely small amount of hydrogen peroxide owing to its high sensitivity. For instance, in the measurement of hydrogen peroxide at such a concentration as to be satisfactorily measured at a measuring sensitivity equivalent to that of 4-aminoantipyrine-phenol type, there are practical problems. That is, a sample must be taken in an extremely small amount (for example, not more than 10 .mu.l), and therefore pipeting error becomes larger, resulting in variation of the measured values. In addition, a sample must first be diluted so as to lessen the errors in sampling. Accordingly, if there were available a method which would allow the adjustment of the sensitivity of the triphenyl methane derivative (which has the advantages, for instance, of a coloring wavelength on the long wavelength side and suffers almost no color fading with a lapse of time after color development over a wide range of around 5,000 on the lower side up to 100,000 or more on the high sensitivity upper side, its application would be greatly widened and advantages, unexpected in the conventional coloring reagents, would be anticipated.